Cleaning systems for Heating Ventilation and Air Conditioning is important to ensure good indoor air quality. The outbreak of their cleaning is based on a visual inspection does not take into account the content molds, which have effects on the respiratory system. This research aims at providing a mold content of the assessment methodology to help building managers. Five dust generations were made in an exposition chamber mimicking a HVAC duct system. A modified 37-mm cassette with a pre-weighed PVC filter was used to collect the settled dust at a flow rate of 15L/min. Particles recovery percentages collected by the cassettes and those deposited on the filters were calculated for 54 samples. Ten other generations were performed with dust using different levels of mold spores. Sixty samples were analyzed with four methods : culture on Malt Extract Agar, direct microscopic spores count (DMSC), Beta-N-Acetylhexosaminidase assay (NAHA) and 18S-q-PCR assay. The detection limit (DL), replicability, repeatability, the number of spores and correlation coefficient (r) were determined. The recovery percentages were greater than 84%. According methods, the median concentration of spores/100 cm² ranged from 10,000 to 815,000. The DL varies depending on the method from 120 to 218,000 spores/100 cm² and from -0.08 to 0.83. Replicability and repeatability were 1% and 1% for PCR, 5% and 10% for DMSC, 6% and 11% for NAHA, 12% and 11% for culture. The sampling method showed excellent dust collection efficiency. The PCR method is recommended for fungal evaluation of ventilation systems. A field validation is underway.